1. Field of the Invention
The invention concerns a process and kit for cloning of DNA fragments as for example PCR amplification products which cannot be cloned with plasmids because of their size and which are too small to be cloned in cosmids. For this purpose modified cosmid vectors were constructed which are suitable for cloning of DNA amplification products and DNA fragments of a certain size.
2. Description of Related Art
Several state of the art methods for cloning of heterologous DNA are known. The simplest method is the use of plasmid vectors which contain a suitable replication origin, an antibiotic resistance gene as a selection marker as well as at least one suitable restriction site. If heterologous DNA fragments exceeding 10 kb are however inserted in conventional plasmids such as pUC or other pBR derivatives this may lead to structures which cannot, as experience has shown, be cloned without deletions in recombination deficient host cells. The disadvantage of this method is the fact that only DNA fragments of a limited maximum size of approximately 10 kb can be cloned efficiently.
Cloning of larger fragments is, according to the state of the art, performed with vectors and based on the reproduction cycle of the bacteriophage lambda. Here, the lambda DNA present in a linear form is prepared first. Then an internal fragment, the so-called stuffer, is removed with a suitable restriction enzyme digestion. The DNA to be cloned is inserted instead of the stuffer between the vector arms (e.g. lambda-gt10; lambda-gt11) by one ligation step. Subsequently, the resulting DNA is incubated with a phage particle extract which was prepared before. With this so-called in vitro packaging reaction infectious phage particles are produced which can be used for infecting appropriate host bacteria. With this method fragments of up to 20 kb can be cloned (Winnacker, E.-L., In: From Genes to Clones, VCH Weinheim (1987) Winnacker, E.-L (ed), pp 154-167); Rimm, D. L. et al. (1980), Gene 12, 301-309).
The disadvantage of this method is the fact that the DNA cloned as described above always remains inserted in the genome of the lambda phage. The lambda genome itself contains genes that code for lytic functions and thus lead to lysis of the bacteria cells so that the so-called plaques are continuously formed because of a certain infection. Compared to the reproduction and preparation of plasmids the reproduction and preparation of lambda DNA is therefore much more cost- and time-intensive. For this reason lambda vectors are, in practice, only used for gene banks but not for cloning of special DNA fragments.
Furthermore, cosmid vectors suitable for cloning of particularly large DNA fragments are known. These are plasmids which additionally contain a cos region. This cos region is derived from the terminal sequences of the genome of the bacteriophage lambda and has an important function in its reproduction cycle: during the replication of the DNA phage concatamers are produced. They are multimers of the phage genome which are arranged in the form of a tandem. During the packaging reaction the concatamers are split at the cos regions by the A-protein of the lambda phage. From this result single linear phage genomes with cohesive ends which are subsequently packed in phage particles.
The DNA fragments to be cloned and the linearized cosmid vectors are ligated under conditions that would in vitro lead to multimer concatamers. By incubation with a packaging extract monomer molecules result from the concatamers which contain each the fragment to be cloned between 2 cos regions and which are consequently packed in the phage particles of the extract. Subsequently, an infection (also called transduction) in suitable host bacteria is performed. Circular cosmids are produced in the host by the cohesive ends (resulting from the cleavage of the cos regions by the protein A as described above) of the terminal cos sequences. These cosmids contain the fragment to be cloned and can be easily reproduced and prepared due to their plasmid characteristics.
Usual cosmids have a size ranging from 4 to 6 kb since for gene banks the fragments to be cloned must be as large as possible (Maniatis et. al. Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, Laboratory Press, Cold Spring Harbour, N.Y., 1982, p. 45). The size of the fragments to be cloned depends on the size of the cosmid used: with the help of the lambda packaging system DNA molecules of a size ranging from 38 kb to 52 kb are packed. Fragments of this size less the size of the cosmid used can therefore be cloned very efficiently. If a cosmid like for example pHC79 (Hohn and Collins (1980), Gene 11, pp. 291-298) has a size of 6.5 kb fragments from 31.5-45.5 kb can be cloned.
The disadvantage of the cosmids is that only DNA fragments of a certain size can be inserted. There is therefore no state of the art method enabling in a simple way the cloning of fragments that are on the one hand too small for packaging of cosmid vectors and on the other hand too big for cloning with plasmids (without cos regions for in vitro packaging).
Fragments of this size category are produced by PCR amplification reactions for example with the "Expand" Long Template PCR System (Boehringer Mannheim catalogue No. 1681834). This kit contains reagents for a PCR method allowing the amplification of genomic fragments of a maximum size of approximately 30 kb. Before this invention was made direct cloning of the relevant amplification products had however not been possible with the plasmid and cosmid vectors known from the state of the art. If for example one would try to clone a 27 kb PCR product in a 6.5 kb cosmid a 33.5 kb molecule would result; this would however be too small for lambda packaging.